scholarly journals Structural basis of inhibition of a transporter from Staphylococcus aureus, NorC, through a single-domain camelid antibody

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Sushant Kumar ◽  
Arunabh Athreya ◽  
Ashutosh Gulati ◽  
Rahul Mony Nair ◽  
Ithayaraja Mahendran ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Pathogenic Bacteria ◽  
Major Facilitator Superfamily ◽  
Single Domain ◽  
Fluoroquinolone Resistance ◽  
Structural Basis ◽  
Antibody Complex ◽  
Complementarity Determining Regions ◽  
Major Facilitator ◽  
Alternating Access

AbstractTransporters play vital roles in acquiring antimicrobial resistance among pathogenic bacteria. In this study, we report the X-ray structure of NorC, a 14-transmembrane major facilitator superfamily member that is implicated in fluoroquinolone resistance in drug-resistant Staphylococcus aureus strains, at a resolution of 3.6 Å. The NorC structure was determined in complex with a single-domain camelid antibody that interacts at the extracellular face of the transporter and stabilizes it in an outward-open conformation. The complementarity determining regions of the antibody enter and block solvent access to the interior of the vestibule, thereby inhibiting alternating-access. NorC specifically interacts with an organic cation, tetraphenylphosphonium, although it does not demonstrate an ability to transport it. The interaction is compromised in the presence of NorC-antibody complex, consequently establishing a strategy to detect and block NorC and related transporters through the use of single-domain camelid antibodies.

scholarly journals Structural basis of inhibition of a putative drug efflux transporter NorC, through a single-domain camelid antibody

2020 ◽  
Author(s):  
Sushant Kumar ◽  
Arunabh Athreya ◽  
Ashutosh Gulati ◽  
Rahul Mony Nair ◽  
Aravind Penmatsa
Keyword(s):  
Major Facilitator Superfamily ◽  
Single Domain ◽  
Fluoroquinolone Resistance ◽  
Structural Basis ◽  
Drug Efflux ◽  
Major Mechanism ◽  
Antibody Complex ◽  
Complementarity Determining Regions ◽  
Major Facilitator ◽  
Alternating Access

AbstractMulti-drug efflux is a major mechanism of acquiring antimicrobial resistance among superbugs. In this study, we report the X-ray structure of NorC, a 14 transmembrane major facilitator superfamily member that is implicated in fluoroquinolone resistance in drug-resistant Staphylococcus aureus strains, at a resolution of 3.6 Å. The NorC structure was determined in complex with a single-domain camelid antibody that interacts at the extracellular face of the transporter and stabilizes it in an outward-open conformation. The complementarity determining regions of the antibody enter and block solvent access to the interior of the vestibule, thereby inhibiting alternating-access. NorC specifically interacts with an organic cation, tetraphenylphosphonium, although it does not demonstrate an ability to transport it. The interaction is compromised in the presence of NorC-antibody complex, consequently establishing a strategy to detect and block NorC and related efflux pumps through the use of single- domain camelid antibodies.

scholarly journals Novel Chromosomally Encoded Multidrug Efflux Transporter MdeA in Staphylococcus aureus

2004 ◽  
Vol 48 (3) ◽  
pp. 909-917 ◽  
Author(s):  
Jianzhong Huang ◽  
Paul W. O'Toole ◽  
Wei Shen ◽  
Heather Amrine-Madsen ◽  
Xinhe Jiang ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Pathogenic Bacteria ◽  
Major Facilitator Superfamily ◽  
Multidrug Efflux ◽  
Expression Library ◽  
Reading Frame ◽  
Transmembrane Segments ◽  
Antibiotic Compounds ◽  
Major Facilitator ◽  
Spontaneous Mutants

ABSTRACT Antibiotic efflux is an important mechanism of resistance in pathogenic bacteria. Here we describe the identification and characterization of a novel chromosomally encoded multidrug resistance efflux protein in Staphylococcus aureus, MdeA (multidrug efflux A). MdeA was identified from screening an S. aureus open reading frame expression library for resistance to antibiotic compounds. When overexpressed, MdeA confers resistance on S. aureus to a range of quaternary ammonium compounds and antibiotics, but not fluoroquinolones. MdeA is a 52-kDa protein with 14 predicted transmembrane segments. It belongs to the major facilitator superfamily and is most closely related, among known efflux proteins, to LmrB of Bacillus subtilis and EmrB of Escherichia coli. Overexpression of mdeA in S. aureus reduced ethidium bromide uptake and enhanced its efflux, which could be inhibited by reserpine and abolished by an uncoupler. The mdeA promoter was identified by primer extension. Spontaneous mutants selected for increased resistance to an MdeA substrate had undergone mutations in the promoter for mdeA, and their mdeA transcription levels were increased by as much as 15-fold. The mdeA gene was present in the genomes of all six strains of S. aureus examined. Uncharacterized homologs of MdeA were present elsewhere in the S. aureus genome, but their overexpression did not mediate resistance to the antibacterials tested. However, MdeA homologs were identified in other bacteria, including Bacillus anthracis, some of which were shown to be functional orthologs of MdeA.

scholarly journals Competition between protons and substrate for binding to the major facilitator superfamily multidrug/H+ antiporter MdtM

2021 ◽  
Vol 2 ◽  
Author(s):  
Christopher J. Law
Keyword(s):  
Escherichia Coli ◽  
General Feature ◽  
Pathogenic Bacteria ◽  
Major Facilitator Superfamily ◽  
Electrochemical Gradient ◽  
Multidrug Efflux ◽  
Protein Fluorescence ◽  
Ph Values ◽  
Major Facilitator ◽  
Intrinsic Protein Fluorescence

Abstract Proton electrochemical gradient-driven multidrug efflux activity of representatives of the major facilitator superfamily (MFS) of secondary active transporters contributes to antimicrobial resistance of pathogenic bacteria. Integral to the mechanism of these transporters is a proposed competition between substrate and protons for the binding site of the protein. The current work investigated the competition between protons and antimicrobial substrate for binding to the Escherichia coli MFS multidrug/H+ antiporter MdtM by measuring the quench of intrinsic protein fluorescence upon titration of substrate tetraphenylphosphonium into a solution of purified MdtM over a range of pH values between pH 8.8 and 5.9. The results, which revealed that protons inhibit binding of substrate to MdtM in a competitive manner, are consistent with those reported in a study on the related MFS multidrug/H+ antiporter MdfA and provide further evidence that competition for binding between substrate and protons is a general feature of secondary multidrug efflux.

scholarly journals It takes two to tango: The dance of the permease

2019 ◽  
Vol 151 (7) ◽  
pp. 878-886 ◽  
Author(s):  
H. Ronald Kaback ◽  
Lan Guan
Keyword(s):  
Binding Sites ◽  
Cytoplasmic Membrane ◽  
Major Facilitator Superfamily ◽  
Internal Cavity ◽  
Lactose Permease ◽  
Physiological Conditions ◽  
Membrane Transport Proteins ◽  
Major Facilitator ◽  
Alternating Access ◽  
Insight Into

The lactose permease (LacY) of Escherichia coli is the prototype of the major facilitator superfamily, one of the largest families of membrane transport proteins. Structurally, two pseudo-symmetrical six-helix bundles surround a large internal aqueous cavity. Single binding sites for galactoside and H+ are positioned at the approximate center of LacY halfway through the membrane at the apex of the internal cavity. These features enable LacY to function by an alternating-access mechanism that can catalyze galactoside/H+ symport in either direction across the cytoplasmic membrane. The H+-binding site is fully protonated under physiological conditions, and subsequent sugar binding causes transition of the ternary complex to an occluded intermediate that can open to either side of the membrane. We review the structural and functional evidence that has provided new insight into the mechanism by which LacY achieves active transport against a concentration gradient.

scholarly journals Structural basis for oligomerization of the prokaryotic peptide transporter PepTSo2

2019 ◽  
Vol 75 (5) ◽  
pp. 348-358 ◽  
Author(s):  
Reina Nagamura ◽  
Masahiro Fukuda ◽  
Akihiro Kawamoto ◽  
Kyoko Matoba ◽  
Naoshi Dohmae ◽  
...  
Keyword(s):  
Shewanella Oneidensis ◽  
Processing System ◽  
Major Facilitator Superfamily ◽  
Peptide Transporter ◽  
Structural Basis ◽  
Cubic Phase ◽  
Tetrameric Structure ◽  
Automated Data Processing ◽  
Major Facilitator ◽  
Extracellular Environment

Proton-dependent oligopeptide transporters (POTs) belong to the major facilitator superfamily (MFS) and transport dipeptides and tripeptides from the extracellular environment into the target cell. The human POTs PepT1 and PepT2 are also involved in the absorption of various orally ingested drugs. Previously reported structures revealed that the bacterial POTs possess 14 helices, of which H1–H6 and H7–H12 constitute the typical MFS fold and the residual two helices are involved in the cytoplasmic linker. PepTSo2 from Shewanella oneidensis is a unique POT which reportedly assembles as a 200 kDa tetramer. Although the previously reported structures suggested the importance of H12 for tetramer formation, the structural basis for the PepTSo2-specific oligomerization remains unclear owing to the lack of a high-resolution tetrameric structure. In this study, the expression and purification conditions for tetrameric PepTSo2 were optimized. A single-particle cryo-EM analysis revealed the tetrameric structure of PepTSo2 incorporated into Salipro nanoparticles at 4.1 Å resolution. Furthermore, a combination of lipidic cubic phase (LCP) crystallization and an automated data-processing system for multiple microcrystals enabled crystal structures of PepTSo2 to be determined at resolutions of 3.5 and 3.9 Å. The present structures in a lipid bilayer revealed the detailed mechanism for the tetrameric assembly of PepTSo2, in which a characteristic extracellular loop (ECL) interacts with two asparagine residues on H12 which were reported to be important for tetramerization and plays an essential role in oligomeric assembly. This study provides valuable insights into the oligomerization mechanism of this MFS-type transporter, which will further pave the way for understanding other oligomeric membrane proteins.

scholarly journals Modulation of antimicrobial efflux pumps of the major facilitator superfamily in Staphylococcus aureus

2018 ◽  
Vol 4 (1) ◽  
pp. 1-18 ◽  
Author(s):  
Manjusha Lekshmi ◽  
◽  
Parvathi Ammini ◽  
Jones Adjei ◽  
Leslie M. Sanford ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Efflux Pumps ◽  
Major Facilitator Superfamily ◽  
Major Facilitator

scholarly journals LmrS Is a Multidrug Efflux Pump of the Major Facilitator Superfamily from Staphylococcus aureus

2010 ◽  
Vol 54 (12) ◽  
pp. 5406-5412 ◽  
Author(s):  
Jody L. Floyd ◽  
Kenneth P. Smith ◽  
Sanath H. Kumar ◽  
Jared T. Floyd ◽  
Manuel F. Varela
Keyword(s):  
Staphylococcus Aureus ◽  
Efflux Pump ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
Relative Increase ◽  
Major Facilitator Superfamily ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Major Facilitator ◽  
Lincomycin Resistance

ABSTRACT A multidrug efflux pump designated LmrS (lincomycin resistance protein of Staphylococcus aureus), belonging to the major facilitator superfamily (MFS) of transporters, was cloned, and the role of LmrS in antimicrobial efflux was evaluated. The highest relative increase in MIC, 16-fold, was observed for linezolid and tetraphenylphosphonium chloride (TPCL), followed by an 8-fold increase for sodium dodecyl sulfate (SDS), trimethoprim, and chloramphenicol. LmrS has 14 predicted membrane-spanning domains and is homologous to putative lincomycin resistance proteins of Bacillus spp., Lactobacillus spp., and Listeria spp.

scholarly journals Function, Structure, and Evolution of the Major Facilitator Superfamily: The LacY Manifesto

2014 ◽  
Vol 2014 ◽  
pp. 1-20 ◽  
Author(s):  
M. Gregor Madej
Keyword(s):  
Structural Information ◽  
Large Body ◽  
Major Facilitator Superfamily ◽  
Lactose Permease ◽  
Test Bed ◽  
X Ray ◽  
X Ray Crystallography ◽  
Major Facilitator ◽  
Mfs Transporters ◽  
Alternating Access

The major facilitator superfamily (MFS) is a diverse group of secondary transporters with members found in all kingdoms of life. A paradigm for MFS is the lactose permease (LacY) of Escherichia coli, which couples the stoichiometric translocation of a galactopyranoside and an H+ across the cytoplasmic membrane. LacY has been the test bed for the development of many methods applied for the analysis of transport proteins. X-ray structures of an inward-facing conformation and the most recent structure of an almost occluded conformation confirm many conclusions from previous studies. Although structure models are critical, they are insufficient to explain the catalysis of transport. The clues to understanding transport are based on the principles of enzyme kinetics. Secondary transport is a dynamic process—static snapshots of X-ray crystallography describe it only partially. However, without structural information, the underlying chemistry is virtually impossible to conclude. A large body of biochemical/biophysical data derived from systematic studies of site-directed mutants in LacY suggests residues critically involved in the catalysis, and a working model for the symport mechanism that involves alternating access of the binding site is presented. The general concepts derived from the bacterial LacY are examined for their relevance to other MFS transporters.

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